A large body of evidence suggests that bone is a dynamic tissue that can heal spontaneously following injury. However, this regenerative process often fails in the geriatric population contributing to widespread aging- related injuries to the musculoskeletal system. Inhibition of mRNA translation by microRNAs (miRNAs) has emerged as an important regulator of osteogenic signaling pathways. Accordingly, miRNA dysregulation has been implicated in the onset and progression of osteoporosis and osteoarthritis. These findings provide a rational basis for the development of novel bone bioengineering therapies that target specific miRNAs in a spatially and temporally controlled fashion. Among its members, miR-29b-1-3p is considered a mature dominant miRNA species that facilitates osteogenic differentiation of human mesenchymal stem/stromal cells (MSCs). In contrast, little is known about miR-29b-1-5p, the other mature miRNA expressed from the same precursor, which until recently was thought of as a byproduct. We have preliminary evidence that miR-29b-1- 5p is anti-osteogenic and that MSCs isolated from geriatric patients exhibit elevated levels of miR-29b-1-5p compared to adult MSCs, while the expression of miR-29b-1-3p is unchanged. These novel findings suggest a pathogenic role for miR-29b-1-5p in aging-related defects in osteogenesis and bone regeneration. Based on these findings, we hypothesize that the miR-29b-1-5p/miR-29b-1-3p ratio increases with age thereby tilting the scale towards anti-osteogenic characteristics. We further hypothesize that inhibitors of miR-29b-1-5p (anti- miRNA therapy) would be effective in reversing anti-osteogenic effects and promoting bone regeneration with progressing age. In this proposal, we aim to elucidate the molecular basis by which miR-29b-1-5p suppresses osteogenic gene targets and disrupts osteogenic differentiation of MSCs with aging, and to determine the effects of altering miR-29b-1-5p levels on MSC-mediated local bone formation in vivo using novel polymer hydrogels. To address these aims, we will use a variety of molecular cell biology and functional assays prior to employing an established orthotopic bone injury model. These studies will provide preclinical evidence for the therapeutic potential of miR-29b-1-5p inhibitors to promote bone regeneration and also for an effective and improved delivery system for anti-miRNA therapy.